Persistent eosinophilic inflammation is a principal characteristic of asthma, with important physiologic consequences. The intensity of the allergic inflammatory response does not differentiate allergic subjects, with and without asthma. Inflammation in asthma, however, is prolonged. Therefore, signals that switch-off inflammation may be crucial in promoting resolution of allergic inflammation. One putative switch-off factor is the cytokine interleukin-10 [IL-10], which induces eosinophil apoptosis, and suppresses TNF-" production. We have shown that lung expression of IL-10 in allergic asthmatics is virtually absent, in contrast to allergic non-asthmatics, in whom IL-10 levels are increased compared to healthy volunteers. We propose the following Central Hypothesis: IL-10 is a critically important to the resolution of allergic inflammation. A genetically-engineered IL-10 deficient "knockout" (IL-10i) mouse strain offers the opportunity to perform critical mechanistic tests of the role of IL-10 in the allergic inflammatory process. Accordingly, the Specific Aims are to establish: 1) the pattern and timing of development and resolution of inflammation and physiologic changes following allergen sensitization and challenge, and the degree and characteristics of allergic sensitization;2) the mechanisms which underlie the observations of Aim 1;3) the specificity of IL-10 in mediating the anti-inflammatory response after allergen challenge, and 4) the role of TNF-" in enabling the inflammatory response to allergen challenge. These cytokines have direct and demonstrable relevance to human asthma. IL-101mice and control strain animals will undergo allergen sensitization and challenge;allergen specific IgE, differentiation of Th1-Th2 lymphocytes, allergen-driven inflammation, and airway hyperresponsiveness will be key outcome variables. In addition, functional knock-out animals will be produced by administration of neutralizing IL-10 antibody, and will undergo similar studies. This information will provide direct evidence of the role of IL-10 in regulating allergic inflammation, and will help to ascertain the relationships between inflammation and airway hyperresponsiveness. PERFORMANCE SITE(S) (organization, city, state) University of Pittsburgh Pittsburgh, Pennsylvania KEY PERSONNEL. See instructions on Page Name William J. Calhoun, M.D. Associate Professor BillT. Ameredes, Ph.D. Research Assistant Professor Timothy Billiar, M.D. Professor and Chair Candace Johnson, Ph.D. Professor of Pharmacology 11. Use continuation pages as needed^ provide Organization University of Pittsburgh Department of Medicine University of Pittsburgh Department of Medicine University of Pittsburgh Department of Surgery University of Pittsburgh Department of Medicine PHS 398 (Rev. 4/98) Page 2 Number pages consecutively the application. Do not use suffixes such as 3a, 3b. the required information in the format shown below. Role on Project Principal Investigator Co-Investigator Consultant Consultant BB at the bottom throughout C:altthiocun, William J. CO Principal Investigator/Program Director (Last, first, middle): Type the name of the principal investigator/program director at the top of each printed page and each continuation page. (For type specifications, see instructions on page 6.) RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description, Performance Sites, and Personnel 2- ~ Table of Contents _3 Detailed Budget for Initial Budget Period Budget for Entire Proposed Period of Support j_ Budgets Pertaining to Consortium/Contractual Arrangements Biographical Sketch-Principal Investigator/Program Director (Notto exceed two pages) Other Biographical Sketches (Notto exceed two pages for each) Other Support Resources lZli?_ Research Plan 19-21 Introduction to Revised Application (Notto exceed 3 pages) Introduction to Supplemental Application (Notto exceed 1page) a. Specific Aims ?!\ /"" 22 b. Background and Significance f. 1 t_I[unreadable]__ n ,[unreadable] [unreadable] c. j- /n n _. X" (Items 3-d: not to exceed 25 pages') <?6-"n c. Preliminary Studies/Progress Report / ''. | ""pound_ d. Research Design andMethods J. V ?4-45_ e. Human Subjects f. Vertebrate Animals 46 g. Literature Cited __lZi5Q_ h. Consortium/Contractual Arrangements i. Consultants 51-52 Checklist __53 *Type density and type size of the entire application must conform to limits provided in instructions on page 6. Appendix (Five collated sets. No page numbering necessary for Appendix.) Check if Number of publications and manuscripts accepted or submitted for publication (notto exceed 10) Appendix is Other items (list): included PHS 398 (Rev. 4/98) (Form Page 3) Page __3._ CC Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b.